A benzofuranone derivative and its application

Abstract

The present invention belongs to the technical field of cell membrane fluorescent probes, and specifically relates to a benzofuranone derivative and its application. The present invention utilizes an N-substituted aminobenzaldehyde structure and 6-hydroxy-benzofuran-3-one condensation to obtain a series of fluorescent probe compounds, and the provided probe compounds can all achieve cell membrane positioning and fluorescence imaging, and the imaging mechanism is that after the probe is combined with the cell membrane, the high viscosity of the cell membrane causes the probe molecule to rotate within the limited, and the fluorescence enhancement is achieved by suppressing the intramolecular torsional charge transfer. Experimental results show that the cell membrane localization specificity and imaging ability of the provided benzofuranone derivatives are jointly affected by the structural characteristics of the fluorescent skeleton of the fluorescent molecule itself and the fatty chain modification, and some of the compounds show the specificity of cell membrane positioning, and no obvious probe endocytosis occurs for a long time.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell membrane fluorescent probes and more specifically relates to a benzofuranone derivative and application thereof. Background Art

[0002] The cell membrane is an elastic semipermeable membrane mainly composed of phospholipids and proteins. Proteases, cytokines and their receptors on the cell membrane initiate cell signal transduction through direct or indirect interactions, and play an important role in regulating the selective transport of molecules and intercellular communication. Accurately locating the cell membrane and real-time monitoring of its dynamic changes not only help us understand the physiological functions of the cell membrane more comprehensively, but also provide new ideas and methods for the diagnosis and treatment of diseases. Small molecule fluorescence has the advantages of high spatial and temporal resolution, high sensitivity, and real-time monitoring of dynamic changes. It has become one of the important methods for cell membrane imaging and provides strong technical support for the research of life sciences and basic medicine.

[0003] So far, a variety of cell membrane small molecule fluorescent probes have been reported, and a few have been commercialized, such as: cell membrane green fluorescent probe BioTracker 490Green (SCT106, Merck), cell membrane green fluorescent probe DiO (CAS28462-56-8); cell membrane red fluorescent probe Cell Tracker CM-DiI (CAS: 180854-97-1), etc. From the perspective of probe design, although different probes use different fluorescent skeletons, they all follow similar membrane localization strategies, such as: introducing long-chain fatty groups to strengthen the interaction between the probe and the hydrophobic phospholipid bilayer; introducing positive charges (such as quaternary ammonium salts) to strengthen the electrostatic interaction between the molecule and the negatively charged phosphate group of phospholipids. However, due to the introduction of long-chain fatty groups, the introduction of probes will cause water solubility problems, and the probes are prone to aggregation, leading to endocytosis and entering the cell, resulting in nonspecific fluorescence signals in the cytoplasm. In addition, the introduction of strong positive charges can easily lead to nonspecific interactions between the probe and proteins on the culture medium and cells, which can also lead to endocytosis of the probe and cause nonspecific fluorescence in the cytoplasm. It is a common phenomenon that the design of membrane probes with positive charge + long-chain fatty groups leads to probe internalization and thus produces non-specific cytoplasmic fluorescence. For example, the product BioTracker 490Green (Cat. No. SCT106, https: / / www.sigmaaldrich.cn / CN / en / product / mm / sct106) from Merck can show obvious cytoplasmic fluorescence signals when performing cell membrane imaging. Summary of the invention

[0004] Existing cell membrane small molecule fluorescent probes generally achieve the purpose of cell membrane localization by introducing long-chain fatty groups and positive charges to strengthen the interaction between the probe and the hydrophobic and negatively charged phospholipid bilayer. However, such probes are also prone to enter cells through endocytosis, thereby causing non-specific cytoplasmic fluorescence. When cell membrane imaging is performed, obvious cytoplasmic fluorescence signals can be seen. The technical problem to be solved by the present invention is to overcome this defect and deficiency and provide the use of benzofuranone derivatives in the preparation of cell membrane fluorescent probes.

[0005] The object of the present invention is to provide a benzofuranone derivative, which is a newly synthesized compound. The overall charge property of this type of compound is close to neutral, and cell membrane localization and fluorescence imaging can be achieved. Some compounds show specificity of cell membrane localization and no obvious probe endocytosis occurs for a long time.

[0006] Another object of the present invention is to provide a cell membrane fluorescent probe.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention protects the use of benzofuranone derivatives in the preparation of cell membrane fluorescent probes, and the structure of the benzofuranone derivatives is shown in formula (I):

[0009]

[0010] Wherein, in formula (I), R 1 Selected from C 1～12 Alkyl or hydrogen; R 2 , R 3 All selected from C 1～6 alkyl.

[0011] Preferably, in formula (I), the R 1 Selected from C 1～8 Alkyl or hydrogen; R 2 , R 3 All selected from C 1～2 Alkyl, and R2=R3.

[0012] More preferably, in formula (I), the R 1 Selected from C 3～8 Alkyl or hydrogen; R 2 , R 3 Selected from C 1～2 Alkyl, and R2=R3.

[0013] More preferably, in formula (I), the R 1 is selected from propyl, hexyl, octyl or hydrogen; R 2 , R 3are all selected from methyl or ethyl, and R2=R3.

[0014] More preferably, in the above application, the benzofuranone derivative has any of the following structures:

[0015]

[0016] The present invention protects a benzofuranone derivative, the structure of which is shown in formula (I):

[0017]

[0018] Wherein, in formula (I), R 1 Selected from C 1～12 Alkyl or hydrogen; R 2 , R 3 All selected from C 1～6 Alkyl; and does not include the following compounds:

[0019] In view of the above problems existing in the current membrane probes, the present invention innovatively proposes to use a class of electrically neutral fluorescent skeletons to realize the preparation of membrane probes, and at the same time, the membrane localization ability is regulated by introducing fatty chains of different lengths. Benzofuranone is a functional group commonly contained in a variety of natural bioactive molecules. The inventor team noticed that it contains an active methylene structure and is an excellent raw material for constructing donor-π-acceptor (D-π-A) type fluorescent molecules through Knoevenagel condensation reaction. Based on the above findings, the applicant condensed 6-hydroxy-benzofuran-3-one with an N-substituted aminobenzaldehyde structure to obtain a series of fluorescent probe compounds. The obtained probes can all realize cell membrane imaging, and the imaging mechanism is that after the probe is combined with the cell membrane, the high viscosity of the cell membrane causes the rotation of the probe molecule to be limited, and the fluorescence enhancement is achieved by inhibiting the intramolecular torsional charge transfer (TICT). The experimental results show that the benzofuranone derivatives provided by the present invention can realize cell membrane localization and fluorescence imaging, and some probe compounds show the specificity of cell membrane localization, and no obvious probe endocytosis occurs for a long time. The molecular design of this type of probe compound is significantly different from the previously used strategy of positive charge + fat chain modification. Its overall charge property is close to neutral, and the compound without fat chain modification has the ability to localize on the cell membrane. The inventor team found that the length of the fat chain has a significant effect on the specificity of cell membrane localization and cell membrane imaging, but the change pattern is relatively complex, which shows that the cell membrane localization specificity and imaging ability of the benzofuranone derivatives provided by the present invention are jointly affected by the structural characteristics of the fluorescent skeleton of the fluorescent molecule itself and the fat chain modification.

[0020] Preferably, it is characterized in that, in formula (I), the R1 Selected from C 1～8 Alkyl or hydrogen; R 2 , R 3 All selected from C 1～2 Alkyl, and R2=R3.

[0021] More preferably, in formula (I), the R 1 Selected from C 3～8 Alkyl or hydrogen; R 2 , R 3 All selected from C 1～2 Alkyl, and R2=R3.

[0022] More preferably, in (I), the R 1 is selected from propyl, hexyl, octyl or hydrogen; R 2 , R 3 are all selected from methyl or ethyl, and R2=R3.

[0023] More preferably, the benzofuranone derivative has any of the following structures:

[0024]

[0025] The present invention also protects a cell membrane fluorescent probe, which contains any one or more of any of the benzofuranone derivatives.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention uses 6-hydroxy-benzofuran-3-one and aromatic aldehyde as raw materials to prepare two fluorescent molecules (CMO-1 and CMO-5) through Knoevenagel condensation reaction, and further uses this type of fluorescent molecules to construct fatty chain-modified probes (CMO-2, CMO-3, CMO-4, CMO-6, CMO-7, CMO-8), among which CMO-1, CMO-2, CMO-3, CMO-4, CMO-6, CMO-7, and CMO-8 are newly synthesized compounds, and the application of CMO-5 in the preparation of cell membrane fluorescent probes is discovered for the first time.

[0028] (2) The probe compounds provided by the present invention can achieve cell membrane localization and fluorescence imaging. The imaging mechanism is that after the probe binds to the cell membrane, the high viscosity of the cell membrane leads to limited rotation of the probe molecule, and fluorescence enhancement is achieved by inhibiting the intramolecular torsional charge transfer. The experimental results show that the cell membrane localization specificity and imaging ability of the provided benzofuranone derivatives are jointly affected by the structural characteristics of the fluorescent skeleton of the fluorescent molecule itself and the fatty chain modification. Some of the compounds show specificity of cell membrane localization, and no obvious probe endocytosis occurs for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the 1H-NMR spectrum of probe CMO-2.

[0030] Figure 2 13C-NMR spectrum of probe CMO-2.

[0031] Figure 3 This is the 1H-NMR spectrum of probe CMO-8.

[0032] Figure 4 This is the 13C-NMR spectrum of probe CMO-8.

[0033] Figure 5 (a) Absorption spectrum of probe CMO-1 / 2 / 3 / 4; (b) Fluorescence spectrum of probe CMO-1 / 2 / 3 / 4; (c) Absorption spectrum of probe CMO-5 / 6 / 7 / 8; (d) Fluorescence spectrum of probe CMO-5 / 6 / 7 / 8.

[0034] Figure 6 The data statistics of CMO-1 / 2 / 3 / 4 for cell membrane fluorescence imaging; the first picture from the left in each group is a fluorescence image, the second is a bright field image, the third is a superimposed image of fluorescence and bright field, and the rightmost picture is a statistical curve of fluorescence intensity changes along the white selection line of the fluorescence image (from left to right); the black line scale in the figure is 20μm.

[0035] Figure 7 The data statistics of CMO-5 / 6 / 7 / 8 for cell membrane fluorescence imaging. In each group, the first image from the left is a fluorescence image, the second image is a bright field image, the third image is a superimposed image of fluorescence and bright field, and the rightmost image is a data statistics of the fluorescence intensity change curve along the white selection line of the fluorescence image (from left to right); the black line scale in the figure is 20μm.

[0036] Figure 8 The data statistics of fluorescence imaging after CMO-2 and HeLa cells were co-incubated for 0.5h, 2h, and 5h, respectively; the first picture from the left of each group is a fluorescence image, the second is a bright field image, the third is the image after the fluorescence and bright field are superimposed, and the rightmost picture is a data statistics of the fluorescence intensity change curve along the white selection line of the fluorescence image (from left to right); the black line scale in the figure is 20μm.

[0037] Fig. 9The data statistics of fluorescence imaging after the CMO-8 probe was co-incubated with HeLa cells for 0.5h, 2h, and 5h, respectively; the first picture from the left of each group is a fluorescence image, the second is a bright field image, the third is the image after the fluorescence and bright field are superimposed, and the rightmost picture is a data statistics of the fluorescence intensity change curve along the white selection line of the fluorescence image (from left to right); the black line scale in the figure is 20μm.

[0038] Fig.10 It is a statistical diagram of the toxicity test results of each probe on HeLa. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Example 1 Preparation of CMO-1 and CMO-5 probes

[0042] The synthetic routes of CMO-1 and CMO-5 are as follows:

[0043]

[0044] The preparation method of CMO-1 specifically includes the following steps: weigh 6-hydroxy-benzofuran-3-one (1, 60 mg, 0.40 mmol) in a pressure-resistant reaction tube and dissolve it with 3 mL of glacial acetic acid. Add p-dimethylaminobenzaldehyde (2, 71.9 mg, 0.48 mmol) to the solution, and then add 2 drops of concentrated hydrochloric acid. Place the reaction solution in an oil bath and stir the reaction at 60°C for 4 to 6 hours. After the reaction is completed, cool to room temperature and concentrate under reduced pressure to obtain a crude product. The obtained crude product is purified by silica gel column chromatography (dichloromethane: methanol = 25: 1), and the solid product CMO-1 (92.4 mg, yield 82.2%) is obtained after removing the organic solvent by rotary evaporation.

[0045] (Z)-2-(4-(Dimethylamino)benzylidene)-6-hydroxybenzofuran-3(2H)-one (CMO-1): red solid, 92.4 mg, yield 82.2%. 1 H NMR (400MHz, DMSO-d6) δ11.05(s,1H),7.78(d,J=8.3Hz,2H),7.57(d,J=8.3Hz ,1H),6.80(s,1H),6.77(d,J=3.0Hz,2H),6.69(d,J=9.3Hz,2H),3.00(s,6H).13 C NMR(101MHz,DMSO-d6)δ180.74,166.99,165.68,151.00,145.01,132.92,125.41,119.1 4,113.59,112.66,112.55,112.44,111.98,111.88,98.47,98.36.HRMS(ESI)m / z:[M+H] + Calcd for C 17 H 16 NO3 282.1130; Found 282.1122.

[0046] The preparation method of CMO-5 specifically includes the following steps: weigh 6-hydroxy-benzofuran-3-one (1, 60 mg, 0.40 mmol) in a pressure-resistant reaction tube and dissolve it with 3 mL of glacial acetic acid. Add p-diethylaminobenzaldehyde (4, 85.0 mg, 0.48 mmol) to the solution, and then add 2 drops of trifluoroacetic acid. Place the reaction solution in an oil bath and stir the reaction at 60°C for 4 to 6 hours. After the reaction is completed, cool to room temperature and concentrate under reduced pressure to obtain a crude product. The obtained crude product is purified by silica gel column chromatography (dichloromethane: methanol = 25: 1), and the solid product CMO-5 (86.6 mg, yield 70.1%) is obtained after removing the organic solvent by rotary evaporation.

[0047] (Z)-2-(4-(Diethylamino)benzylidene)-6-hydroxybenzofuran-3(2H)-one (CMO-5): yellow-brown solid, 86.6 mg, yield 70.1%. 1 H NMR (400MHz, DMSO-d6) δ10.92(s,1H),7.76(d,J=8.7Hz,2H),7.56(d,J=8.4Hz,1H),6.75( dd,J=5.5,3.5Hz,3H),6.68(d,J=6.9Hz,2H),3.41(q,J=7.0Hz,4H),1.12(t,J=7.0Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ181.13,167.67,166.63,132.71,125.81,113.18,113.07,112.86,98.71,98.59,10.80.HRMS(ESI)m / z:[M+H] + Calcdfor C 19 H 20 NO3310.1443; Found 310.1436.

[0048] Example 2 Preparation of CMO-2, CMO-3, CMO-4, CMO-6, CMO-7, CMO-8 probes CMO-2, CMO-3, CMO-4 are synthesized using CMO-1 as raw material, and probes CMO-6, CMO-7, CMO-8 are synthesized using CMO-5 as raw material. CMO-1 and CMO-5 are reacted with different brominated (or iodinated) alkanes respectively. The reaction solvent can be any one of acetonitrile (ACN), dioxane, tetrahydrofuran or other solvents that can react. Any one of Na2CO3, K2CO3, NaOH, Cs2CO3 or other inorganic bases are added to the reaction system to catalyze the reaction. The reaction is carried out smoothly under heating and stirring at 50-80°C, and increasing the temperature can accelerate the reaction. Their synthetic routes are as follows:

[0049]

[0050] The preparation method of CMO-2 to CMO-4 specifically includes the following steps: weigh CMO-1 (30.9 mg, 0.11 mmol) and anhydrous K2CO3 (19.2 mg, 0.14 mmol) in a pressure tube, dissolve with 3 mL of acetonitrile, and then add the corresponding halogenated alkane (0.21 mmol) 1-iodopropane / 1-iodohexane / 1-bromooctane. The reaction solution is heated to 80°C in an oil bath, and the reaction is stirred for 3.5 hours to complete the reaction. After the reaction is completed, it is cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The obtained crude product is purified by silica gel column chromatography (dichloromethane: methanol = 25: 1), and the target probe compounds CMO-2, CMO-3 and CMO-4 are obtained after removing the organic solvent by rotary evaporation.

[0051] (Z)-2-(4-(dimethylamino)benzylidene)-6-propoxybenzofuran-3(2H)-one (CMO-2): orange solid, 23.8 mg, yield 65.0%. 1 H NMR(400MHz, DMSO-d6)δ7.82(d,2H),7.62(dd,J=8.6,2.1Hz,1H),7.07(s,1H),6.87–6.69(m,4H),4. 09(td,J=6.5,2.1Hz,2H),3.01(s,J=2.1Hz,6H),1.77(q,J=7.1Hz,2H),0.99(td,J=7.5,2.1Hz,3H). 13C NMR(101MHz,DMSO-d6)δ180.80,167.00,166.02,151.14,144.91,133.07,124.97,119.01,114.52,113.13 ,113.03,112.58,112.48,111.97,111.87,97.31,97.21,70.09,21.78,10.29,10.22.HRMS(ESI)m / z:[M+H] + Calcd for C 20 H 22 NO3324.1599; Found 324.1593.CMO-2 13 C-NMR spectrum see Figure 1 ,That 1 H-NMR spectrum see Figure 2 .

[0052] (Z)-2-(4-(Dimethylamino)benzylidene)-6-(hexyloxy)benzofuran-3(2H)-one (CMO-3): orange solid, 20.6 mg, yield 51.4%. 1 H NMR(400MHz, DMSO-d6)δ7.83–7.78(d,2H),7.62(d,1H),7.07(s,J=3.5Hz,1H),6.80–6.75(d, 4H),4.13–4.06(s,2H),3.00(s,6H),1.76–1.68(s,2H),1.48–1.12(m,9H),0.90–0.84(s,4H). 13 C NMR(101MHz,DMSO-d6)δ180.77,166.99,166.01,151.13,144.90,133.04,124.93,119.00,114.49,113.09,112.9 9,112.57,112.48,111.95,111.86,97.29,97.19,68.66,30.90,28.33,25.06,22.04,13.90.HRMS(ESI)m / z:[M+H] + Calcd for C 23 H 28 NO3 366.2069; Found 366.2062.

[0053] (Z)-2-(4-(Dimethylamino)benzylidene)-6-(octyloxy)benzofuran-3(2H)-one (CMO-4): orange solid, 40 mg, yield 92.1%. 1H NMR(400MHz, DMSO-d6)δ7.81(d,J=8.4Hz,2H),7.61(d,J=8.5Hz,1H),7.07(s,1H),6.81–6.72(m,4H),4.1 1(s,2H),3.01(s,6H),1.74(s,2H),1.39(d,J=7.5Hz,2H),1.18(m,J=24.7Hz,8H),0.85(t,J=6.3Hz,4H). 13 C NMR (101MHz, CDCl3) δ182.66,167.83,166.47,151.23,146.06,133.38,13 3.35,125.47,120.33,115.53,113.94,113.84,112.29,112.18,112.10,11 1.99,97.05,96.93,69.13,68.97,68.82,40.27,40.20,40.14,31.92,29. 81,29.41,29.33,29.11,26.08,22.77,14.24,14.17.HRMS(ESI)m / z:[M+H] + Calcd for C 25 H 32 NO3 394.2382; Found 394.2367.

[0054] The preparation method of CMO-6 to CMO-8 specifically includes the following steps: weigh CMO-5 (34.0 mg, 0.11 mmol) and anhydrous K2CO3 (19.2 mg, 0.14 mmol) in a pressure tube, dissolve with 3 mL of acetonitrile, and then add the corresponding halogenated alkane (0.21 mmol) 1-iodopropane / 1-iodohexane / 1-bromooctane. The reaction solution is heated to 80°C in an oil bath, and the reaction is stirred for 3.5 hours to complete the reaction. After the reaction is completed, it is cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The obtained crude product is purified by silica gel column chromatography (dichloromethane: methanol = 25: 1), and the target probe compounds CMO-6, CMO-7 and CMO-8 are obtained after removing the organic solvent by rotary evaporation.

[0055] (Z)-2-(4-(Diethylamino)benzylidene)-6-propoxybenzofuran-3(2H)-one (CMO-6): orange oily product, 29.7 mg, yield 76.8%. 1H NMR (400MHz, DMSO-d6) δ7.78(d,J=8.5Hz,2H),7.65–7.58(d,1H),7.04(s,1H),6.80(d,J=8.6Hz,2H),6.75–6.72(d,2 H), 4.08 (t, J = 6.6Hz, 2H), 3.41 (q, J = 7.1Hz, 4H), 1.76 (q, J = 7.0Hz, 2H), 1.11 (t, J = 7.0Hz, 6H), 0.98 (t, J = 7.4Hz, 3H). 13 C NMR (101MHz, DMSO-d6) δ180.66,166.84,165.91,148.67,144.70,133.44,124.90,118.24,114.61,113.28,113.18,112. 47,112.37,111.42,111.33,97.27,97.17,70.04,43.79,43.75,21.76,12.46,12.38,10.27,10.20.HRMS(ESI)m / z:[M+H] + Calcd for C 22 H 26 NO3352.1912; Found 352.1896.

[0056] (Z)-2-(4-(Diethylamino)benzylidene)-6-(hexyloxy)benzofuran-3(2H)-one (CMO-7): orange-yellow solid, 34.5 mg, yield 79.8%. 1 H NMR (400MHz, DMSO-d6) δ7.78(d,J=8.4Hz,2H),7.63–7.57(d,1H),7.05(s,1H),6.79(d,J=8.5Hz,2H),6.77–6.69(s,2H),4.11(t, J=6.6Hz,2H),3.40(d,J=7.1Hz,4H),1.72(t,J=7.3Hz,2H),1.48–1.24(m,6H),1.11(t,J=7.0Hz,6H),0.87(q,J=5.7,5.3Hz,3H). 13C NMR (101MHz, DMSO-d6) δ180.65,166.85,165.91,148.67,144.70,133.44,124.88,118.24,114.59,113.27,113.17,112.49, 112.39,111.42,111.32,97.26,97.16,68.63,43.78,30.90,28.33,25.05,22.02,13.87,12.45,12.37.HRMS(ESI)m / z:[M+H] + Calcd for C 25 H 32 NO3 394.2382; Found 394.2366.

[0057] (Z)-2-(4-(Diethylamino)benzylidene)-6-(octyloxy)benzofuran-3(2H)-one (CMO-8): orange-yellow solid, 37.7 mg, yield 81.4%. 1 H NMR(400MHz,Chloroform-d)δ7.79(d,J=8.5Hz,2H),7.68(d,J=8.3Hz,1H),6.82(s,1H),6.72(d,J=8.8Hz,4H),4.05(t,2H),3 .43(q,J=7.1Hz,4H),1.82(m,J=7.3Hz,2H),1.48(s,2H),1.38–1.28(m,8H),1.22(t,J=8.7,7.0Hz,6H),0.90(t,J=6.5Hz,3H). 13 CNMR(101MHz,DMSO-d6)δ182.63,167.74,166.40,148.94,145.90,133.77,133.69,125.53,125.42,119.53,115.68,114.15 ,112.19,111.58,97.00,68.98,44.66,31.94,29.44,29.35,29.13,26.11,22.79,14.23,12.81,12.74.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 36 NO3 422.2695; Found 422.2690.CMO-8 13 C-NMR spectrum see Figure 3 ,That 1 For details of H-NMR spectrum, see Figure 4 .

[0058] Example 3 Spectral test of probe

[0059] 1. Experimental methods

[0060] Each probe solution was prepared using PBS (pH 7.4) buffer, and 0.2% sodium dodecyl sulfate (SDS) was added to promote dissolution, so that the probe concentration was 20 μM, and spectral testing was performed. Among them, the excitation wavelength of probe CMO-1 / 2 / 3 / 4 was 470nm; the excitation wavelength of probe CMO-5 / 6 / 7 / 8 was 480nm.

[0061] 2. Experimental results

[0062] The results are as follows Figure 5 As shown, Figure 5 (a) Figure 5 (c) The UV-visible absorption spectrum shows that all probes have obvious absorption between 400-550nm, but there are differences in absorbance. The absorption spectrum peaks of CMO-2 / 3 / 4 are around 470nm, and the absorption spectrum peaks of CMO-6 / 7 / 8 are around 480nm. The absorption spectra of the unmodified probes CMO-1 and CMO-5 have a certain blue shift compared to the alkyl-modified probes of the same family. The fluorescence spectrum shows that each probe has fluorescence emission between 500-650nm, with peaks around 570nm. Similarly, the unmodified CMO-1 and CMO-5 have obvious spectral blue shifts compared to other probes ( Figure 5 (b) and Figure 5 (d)). Alkyl groups are considered to have certain electron donating properties, but alkyl groups are generally electrically neutral. Alkyl substitution of hydroxyl groups on the fluorescent skeleton may reduce the energy of D-π-A photon radiation, resulting in a red shift in wavelength.

[0063] Example 4 Cell membrane imaging of probe

[0064] 1. Experimental methods

[0065] Cell culture conditions: HeLa cells of the human cervical cancer cell line were cultured in modified Eagle's medium DMEM containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin mixture (double antibody) and placed in a sterile incubator at 37°C and 5% CO2. During imaging, the cells were seeded on a 35mm confocal culture dish at a density of 150,000 to 200,000 cells per dish. After 24 hours of cell incubation, fluorescent probes were added to the culture medium, and the final concentration of each probe was set to 10 μM. Laser confocal fluorescence imaging was performed 1 hour later, and the fluorescence images were obtained using a Zeiss LSM 800 laser confocal microscope. The imaging excitation condition was set to λ ex =488nm; the light receiving condition is set to λ ex=520~700nm, the color of fluorescence is marked as yellow according to the spectral characteristics of the molecule. Image J was used to analyze the fluorescence imaging photos.

[0066] 2. Experimental results

[0067] The results are as follows Figure 6 and Figure 7 As shown in the figure, all probes can achieve fluorescence imaging of cell membranes after 1 hour of incubation. The mechanism of their imaging is that after the probes bind to the cell membrane, the high viscosity of the cell membrane restricts the rotation of the probe molecules, and the fluorescence enhancement is achieved by inhibiting the intramolecular torsional charge transfer (TICT).

[0068] Surprisingly, the probes CMO-1 and CMO-5 without aliphatic chains also showed obvious cell membrane localization. This indicates that the fluorescent skeleton itself has a certain interaction with the membrane structure, and the aliphatic chain is not necessary for such molecules to achieve membrane localization. However, the introduction of aliphatic chains can make the membrane present a stronger fluorescence signal, indicating that more probes are enriched on the cell membrane. Therefore, the introduction of aliphatic chains can indeed enhance the membrane localization ability of the probe. For N, N-dimethyl substituted fluorescent molecules, the introduction of a short propyl modification (CMO-2) can significantly enhance the fluorescence signal of the membrane, and at the same time does not cause obvious probe endocytosis. When the number of carbon atoms in the aliphatic chain is increased to prepare CMO-3 and CMO-4, their membrane imaging effect is not enhanced accordingly, but instead shows obvious intracellular nonspecific signals. For N, N-diethyl substituted fluorescent molecules, the imaging effect of the long-chain aliphatic group-modified probe CMO-8 is better than that of the unmodified probe (CMO-5) and the short-chain modified probe (CMO-6, CMO-7). It can be seen that due to the different N substituents, the effects of aliphatic chain modification on the imaging effects of the two types of fluorescent skeletons are not consistent. The above results indicate that the structural characteristics of the fluorescent skeleton itself and the fatty chain modification jointly affect the cell membrane imaging ability and membrane localization specificity of the probe.

[0069] The above test results show that the obtained probes can achieve cell membrane fluorescence imaging, and CMO-2 and CMO-8 have better imaging effects. The present invention further investigates the long-term cell membrane localization ability of the two probes, CMO-2 and CMO-8, by adding 4 μM probes to cultured cells and performing fluorescence imaging after incubation for 0.5 h, 2 h and 5 h, respectively.

[0070] The results are as follows Figure 8 and Fig. 9As shown in the figure, both CMO-2 and CMO-8 can achieve cell membrane fluorescence imaging after 0.5 hours of incubation. As the incubation time increases, the signal of CMO-8 on the membrane is significantly enhanced, but the fluorescence enhancement of CMO-2 is not obvious, which indicates that CMO-2 may have a faster interaction with the cell membrane. Importantly, as the incubation time increases, CMO-2 shows a more consistent imaging effect and membrane localization specificity, and 5 hours of incubation did not cause obvious probe endocytosis. For CMO-8, fluorescence signals were observed in the cells after 5 hours of incubation. The above results show that both CMO-2 and CMO-8 can be used to achieve cell membrane-specific fluorescence imaging, but if long-term observation of more than 2 hours is required, CMO-2 has more advantages.

[0071] Example 5 Cell compatibility test of probe

[0072] 1. Experimental methods

[0073] The effect of the probe on cell viability is usually used to evaluate the cell compatibility of the probe. The cell culture conditions refer to the above content. Before the toxicity test, HeLa cells were planted in a 96-well cell culture plate at a density of 8000 cells per well. After 24 hours of cell culture, the culture medium was removed and the cells were continued to be cultured with fresh culture medium containing the probe. After 72 hours, the cell viability was tested using the Ren Tianqing test reagent. The cell viability of the control group without probe treatment was set to 100%.

[0074] 2. Experimental results

[0075] Cell viability Fig.10 As shown. The results showed that among all the eight probes, only probe CMO-5 showed obvious cytotoxicity, with a half-inhibitory concentration of 6.09 μM for cell activity, while the other seven probes showed no obvious cytotoxicity. This shows that these probes are low-toxic at concentrations that meet imaging requirements (usually less than 10 μM), have the potential to achieve long-term cell observation, and will not affect the physiological activity of the cells themselves.

[0076] In summary, the present invention uses 6-hydroxy-benzofuran-3-one and aromatic aldehyde as raw materials, prepares two fluorescent molecules (CMO-1 and CMO-5) through Knoevenagel condensation reaction, and further uses this type of fluorescent molecules to construct fatty chain modified probes (CMO-2, CMO-3, CMO-4, CMO-6, CMO-7, CMO-8), wherein CMO-2 and CMO-6 are propane modified products, CMO-3 and CMO-7 are hexane modified products, and CMO-4 and CMO-8 are octane modified products. The obtained probes can achieve cell membrane localization and fluorescence imaging, wherein the probe compounds CMO-2 and CMO-8 have higher cell membrane localization specificity. In long-term observation, the advantage of CMO-2 is more prominent.

[0077] In addition, the probe compound provided by the present invention is a compound having an aniline skeleton. It is known that aniline has only very weak alkalinity and exhibits a very weak positive charge characteristic under physiological pH (pH 7.4) conditions, which is close to electrical neutrality. Therefore, the overall charge property of the probe compound having an aniline skeleton provided by the present invention is also close to electrical neutrality; and the fluorescent molecules CMO-1 and CMO-5 of the present invention that are not modified with a fat chain have the ability to localize to the cell membrane, which also shows that the molecular design of the probe compound of the present invention is significantly different from the positive charge + fat chain modification strategy used in the past.

[0078] In addition, the inventor team also found that the length of the fat chain has a significant effect on the specificity of cell membrane localization and cell membrane imaging, but the change pattern is relatively complex, which shows that the cell membrane localization specificity and imaging ability of the benzofuranone derivatives provided by the present invention are jointly affected by the structural characteristics of the fluorescent skeleton of the fluorescent molecule itself and the fat chain modification.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of benzofuranone derivatives in the preparation of cell membrane fluorescent probes, characterized in that: The structure of the benzofuranone derivative is shown in formula (I): Among them, in formula (I), R 1 Selected from C 1~12 Alkyl or hydrogen; R 2 , R 3 All selected from C 1~6 alkyl.

2. The application according to claim 1, characterized in that: In formula (I), R 1 Selected from C 1~8 Alkyl or hydrogen; R 2 , R 3 All selected from C 1~2 Alkyl, and R2=R3.

3. The application according to claim 2, characterized in that: In formula (I), R 1 Selected from C 3~8 Alkyl or hydrogen; R 2 , R 3 Selected from C 1~2 Alkyl, and R2=R3.

4. The use according to claim 3, characterized in that: In formula (I), R 1 is selected from propyl, hexyl, octyl or hydrogen; R 2 , R 3 are all selected from methyl or ethyl, and R2=R3.

5. A benzofuranone derivative, characterized in that: The structure of the benzofuranone derivative is shown in formula (I): Among them, in formula (I), R 1 Selected from C 1~12 Alkyl or hydrogen; R 2 , R 3 All selected from C 1~6 Alkyl; and does not include the following compounds: , , , .

6. The benzofuranone derivative according to claim 5, characterized in that: In formula (I), R 1 Selected from C 1~8 Alkyl or hydrogen; R 2 , R 3 All selected from C 1~2 Alkyl, and R2=R3.

7. The benzofuranone derivative according to claim 6, characterized in that: In formula (I), R 1 Selected from C 3~8 Alkyl or hydrogen; R 2 , R 3 All selected from C 1~2 Alkyl, and R2=R3.

8. The benzofuranone derivative according to claim 7, characterized in that: (I), the R 1 is selected from propyl, hexyl, octyl or hydrogen; R 2 , R 3 are all selected from methyl or ethyl, and R2=R3.

9. The benzofuranone derivative according to claim 8, characterized in that: The benzofuranone derivatives have any of the following structures: 。 10. A cell membrane fluorescent probe, characterized in that: The cell membrane fluorescent probe contains any one or more of the benzofuranone derivatives according to any one of claims 5 to 9.

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